Brain imaging in schizophrenia patients reveals excessive loss of gray matter, already visible in young adults at the first psychotic episode. Post mortem brains from schizophrenia (SCZ) patients have decreased numbers of synapses in the prefrontal cortex (PFC), a region involved in executive function and working memory. However, it is not known whether synapse loss results from excessive developmental pruning or why schizophrenia often becomes clinically apparent during adolescence. Dendritic spine density on layer 3 pyramidal neurons is dramatically reduced in SCZ patients, suggesting a synapse and circuit- specific mechanism of vulnerability. As the PFC integrates information from multiple brain regions, defects in pruning could significantly impact synaptic connectivity, cognitive function, and behavior. Our goal is to map the development and refinement of PFC synapses (Aim 1) and to interrogate the functional and behavioral consequences of local and global defects in synaptic pruning (Aims 2?3). We have identified C4A and the classical complement cascade as key mediators of synaptic pruning in the mouse postnatal visual system (Sekar et al., 2016)(Project 2); however, it is unknown if it is also necessary in the cortex and circuits relevant to SCZ and if aberrant pruning affects anatomical and functional connectivity and the behaviors dependent on it. In Aim 2, we will test the hypothesis that over-activation of the complement cascade enhances pruning in the PFC, perturbing anatomical and functional connectivity as well as behavior. We will use global complement KO mice (C1q, C4KO) and novel hC4A-overexpressing mice (Project 2) to ask if early postnatal pruning defects impact cortical connectivity and function later in life. We will also use viral strategies to test if circuit-specific and temporally restricted activation and inhibition of the complement cascade is sufficient for circuit-specific phenotypes. In Aim 3, we will seek to understand how second hits (genetic or environmental) on a background of genetic risk (increased copy number of C4A) combine to impact neural circuit development and behavior. We hypothesize that a second genetic hit (e.g., loss of CSMD1) or environmental hit (immune challenge) might worsen synaptic and behavioral phenotypes in mice over-expressing the human C4A risk allele. To link the central and peripheral effects of C4 expression explored in Projects 1 and 2, we will investigate the potential role of the choroid plexus, a major source of cerebral spinal fluid (CSF), in the regulation of complement and cytokine levels in the brain in Aim 3. This will lead to a better understanding of the cellular and molecular players linking peripheral immune dysregulation to brain dysfunction and may identify novel biomarkers.